High-yield and high-purity isolation of hepatic stellate cells from normal and fibrotic mouse livers.
High-yield and high-purity isolation of hepatic stellate cells from normal and fibrotic mouse livers.
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DOI：
10.1038/nprot.2015.017
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发表时间：
2015-02
期刊：
影响因子：
14.8
通讯作者：
Schwabe RF
中科院分区：
文献类型：
--
作者：
Mederacke I;Dapito DH;Affò S;Uchinami H;Schwabe RF
肝星状细胞（hepatic stellate cells，HSC）是肝脏中主要的纤维化细胞类型。因此，努力了解肝纤维化和开发治疗策略集中在这种细胞类型。最初在大鼠中开发的HSC分离，随后适用于小鼠，从而可以通过遗传方法在转基因小鼠中研究纤维化。然而，小鼠HSC分离通常受到低产量和低纯度的阻碍。在这里，我们提出了一个易于执行的协议，高纯度和高产量的分离静止和激活的HSC在小鼠中，基于逆行链霉蛋白酶-胶原酶灌注的肝脏和随后的密度梯度离心。我们描述了一个可选的附加协议，通过后续的流式细胞术分选从正常和纤维化肝脏中分离超纯HSC，从而提供了一种有效的方法来确定HSC活化过程中的基因表达变化，而不需要细胞培养物或其他细胞的污染。所描述的隔离程序需要大约四个小时才能完成。
Hepatic stellate cells (HSCs) have been identified as the main fibrogenic cell type in the liver. Hence, efforts to understand hepatic fibrogenesis and to develop treatment strategies have focused on this cell type. HSC isolation, originally developed in rats, has subsequently been adapted to mice, allowing to study fibrogenesis by genetic approaches in transgenic mice. However, mouse HSC isolation is commonly hampered by low yield and purity. Here we present an easy-to-perform protocol for high-purity and high-yield isolation of quiescent and activated HSCs in mice, based on retrograde pronase-collagenase perfusion of the liver and subsequent density-gradient centrifugation. We describe an optional add-on protocol for ultrapure HSC isolation from normal and fibrotic livers via subsequent flow-cytometric sorting, thus providing a validated method to determine gene expression changes during HSC activation devoid of cell culture artefacts or contamination with other cells. The described isolation procedure takes approximately four hours to complete.